Membrane fusion

The factors involved in the regulation of biological membrane fusion and models proposed for the molecular mechanism of biomembrane fusion are reviewed. The results obtained in model systems are critically discussed in the light of the known properties of biomembranes and characteristics of biomembrane fusion. Biological membrane fusion is a local-point event; extremely fast, non-leaky, and under strict control. Fusion follows on a local and most probably protein-modulated destabilization, and a transition of the interacting membranes from a bilayer to a non-bilayer lipid structure. The potential role of type II non-bilayer preferring lipids and of proteins in the local destabilization of the membranes is evaluated. Proteins are not only responsible for the mutual recognition of the fusion partners, but are most likely also to be involved in the initiation of biomembrane fusion, by locally producing or activating fusogens, or by acting as fusogens.     

The role of transmembrane domains in membrane fusion

Biological membrane fusion is driven by different types of molecular fusion machines. Most of these proteins are membrane-anchored by single transmembrane domains. SNARE proteins are essential for intracellular membrane fusion along the secretory and endocytic pathway, while various viral fusogens mediate infection of eukaryotic cells by enveloped viruses. Although both types of fusion proteins are evolutionarily quite distant from each other, they do share a number of structural and functional features. Their transmembrane domains are now known to be critical for the fusion reaction. We discuss at which stages they might contribute to bilayer mixing. Received 5 October 2006; received after revision 14 November 2006; accepted 8 January 2007     

Regulation of SNARE fusion machinery by fatty acids

Vesicle fusion is a ubiquitous biological process involved in membrane trafficking and a variety of specialised events such as exocytosis and neurite outgrowth. The energy to drive biological membrane fusion is provided by fusion proteins called SNAREs. Indeed, SNARE proteins play critical roles in neuronal development as well as neurotransmitter and hormone release. SNARE proteins form a very tight alpha-helical bundle that can pull two membranes together, thereby initiating fusion. Whereas a great deal of attention has been paid to partner proteins that can affect SNARE function, recent genetic and biochemical evidence suggests that local lipid environment may be as important in SNARE regulation. Direct lipid modification of SNARE fusion proteins and their regulation by fatty acids following phospholipase action will be discussed here in detail. Our analysis highlights the fact that lipids are not a passive platform in vesicle fusion but intimately regulate SNARE function. Received 20 December 2006; received after revision 6 February 2007; accepted 15 March 2007     

Membrane fusion: the process and its energy suppliers

Membrane fusion constitutes a pivotal process in eukaryotic cell physiology. Both specialized proteins and membrane lipids play key roles in fusion. Here, our current understanding of the mechanism of membrane fusion is reviewed. The focus is on the relatively simple and well-understood proteinaceous fusion machinery of enveloped viruses and the physical properties of lipids that appear to be of great relevance for fusion progression. Recent observations suggest that viral fusion proteins use packed conformational energy and bilayer-destabilizing domains to (i) bring participating membranes into intimate contact, (ii) merge proximal lipid monolayers through highly curved stalk/hemifusion intermediates, and (iii) generate a lipid-containing fusion pore, thereby terminating the fusion process. Received 4 January 2002; received after revision 3 April 2002; accepted 5 April 2002     

Membrane traffic in the secretory pathway

SNARE (SNAP receptor) proteins drive intracellular membrane fusion and contribute specificity to membrane trafficking. The formation of SNAREpins between membranes is spatially and temporally controlled by a network of sequentially acting accessory components. These regulators add an additional layer of specificity, arrest SNAREpin intermediates, lower the energy required for fusion, and couple membrane fusion to triggering signals. The functional activity of some of these regulators determines the plasticity of regulated exocytosis. (Part of a Multi-author Review)     

Molecular machinery mediating vesicle budding,docking and fusion

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs (SNAP receptors) on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.     

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8–10 aa)C signature motif. Structure–function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.     

The ATP-binding cassette family: a structural perspective

The ATP-binding cassette family is one of the largest groupings of membrane proteins, moving allocrites across lipid membranes, using energy from ATP. In bacteria, they reside in the inner membrane and are involved in both uptake and export. In eukaryotes, these transporters reside in the cell’s internal membranes as well as in the plasma membrane and are unidirectional—out of the cytoplasm. The range of substances that these proteins can transport is huge, which makes them interesting for structure–function studies. Moreover, their abundance in nature has made them targets for structural proteomics consortia. There are eight independent structures for ATP-binding cassette transporters, making this one of the best characterised membrane protein families. Our understanding of the mechanism of transport across membranes and membrane protein structure in general has been enhanced by recent developments for this family.     

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

Glycoprotein G of the vesicular stomatitis virus (VSV) is involved in receptor recognition at the host cell surface and then, after endocytosis of the virion, triggers membrane fusion via a low pH-induced structural rearrangement. G is an atypical fusion protein, as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The atomic structures of these two conformations reveal that it is homologous to glycoprotein gB of herpesviruses and that it combines features of the previously characterized class I and class II fusion proteins. Comparison of the structures of G pre- and postfusion states shows a dramatic reorganization of the molecule that is reminiscent of that of paramyxovirus fusion protein F. It also allows identification of conserved key residues that constitute pH-sensitive molecular switches. Besides the similarities with other viral fusion machineries, the fusion properties and structures of G also reveal some striking particularities that invite us to reconsider a few dogmas concerning fusion proteins.     

The membrane domain of vacuolar H<Superscript>+</Superscript>ATPase: a crucial player in neurotransmitter exocytotic release

V-ATPases are multimeric enzymes made of two sectors, a V1 catalytic domain and a V0 membrane domain. They accumulate protons in various intracellular organelles. Acidification of synaptic vesicles by V-ATPase energizes the accumulation of neurotransmitters in these storage organelles and is therefore required for efficient synaptic transmission. In addition to this well-accepted role, functional studies have unraveled additional hidden roles of V0 in neurotransmitter exocytosis that are independent of the transport of protons. V0 interacts with SNAREs and calmodulin, and perturbing these interactions affects neurotransmitter release. Here, we discuss these data in relation with previous results obtained in reconstituted membranes and on yeast vacuole fusion. We propose that V0 could be a sensor of intra-vesicular pH that controls the exocytotic machinery, probably regulating SNARE complex assembly during the synaptic vesicle priming step, and that, during the membrane fusion step, V0 might favor lipid mixing and fusion pore stability.     

Deciphering the BAR code of membrane modulators

The BAR domain is the eponymous domain of the “BAR-domain protein superfamily”, a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.     

Sticky-finger interaction sites on cytosolic lipid-binding proteins?

The cytosolic lipid-binding proteins (cLBPs) comprise a large family of small (14-15 kDa) intracellular proteins involved in the transport of small lipids, including fatty acids and retinoids within cells. Their presumed function is to solubilise, protect from chemical damage and deliver to the correct destination lipids for purposes ranging from energy metabolism (e.g. fatty acids) to signalling, gene activation and cellular differentiation (e.g. retinoids and eicosanoids). It is therefore probable that cLBPs interact directly with cellular components (membranes and/or proteins) to collect and deposit their ligands, and some external features of the different cLBPs may be involved in such interactions and determine which cellular component (integral membrane or cytosolic proteins, or membranes of different lipid compositions or domain structures) with which a given cLBP will interact. Here we have focussed on a previously unrecognised feature of cLBPs which descriminates between those for which there is empiral evidence for direct interaction with membranes, and those which do not. This is a group of bulky hydrophobic amino acid side chains (e.g. tryptophans, phenylalanines, leucines) which project directly into solvent adjacent to the portal of entry and exit of the lipid ligands. Such side chains are usually found internal to proteins, but are common at sites of protein:protein or protein:membrane interactions. These 'sticky fingers' could therefore be critical to the nature and specificity of the interactions cLBPs undergo in the web of cross-traffic in lipid movements within cells.     

Functional significance of the lipid-protein interface in photosynthetic membranes

The functional significance of the lipid-protein interface in photosynthetic membranes, mainly in thylakoids, is reviewed with emphasis on membrane structure and dynamics. The lipid-protein interface is identified primarily by the restricted molecular dynamics of its lipids as compared with the dynamics in the bulk lipid phase of the membrane. In a broad sense, lipid-protein interfaces comprise solvation shell lipids that are weakly associated with the hydrophobic surface of transmembrane proteins but also include lipids that are strongly and specifically bound to membrane proteins or protein assemblies. The relation between protein-associated lipids and the overall fluidity of the thylakoid membrane is discussed. Spin label electron paramagnetic resonance spectroscopy has been identified as the technique of choice to characterize the protein solvation shell in its highly dynamic nature; biochemical and direct structural methods have revealed an increasing number of protein-bound lipids. The structural and functional roles of these protein-bound lipids are mustered, but in most cases they remain to be determined. As suggested by recent data, the interaction of the non-bilayer-forming lipid, monogalactosyldyacilglycerol (MGDG), with the main light-harvesting chlorophyll a/b-binding protein complexes of photosystem-II (LHCII), the most abundant lipid and membrane protein components on earth, play multiple structural and functional roles in developing and mature thylakoid membranes. A brief outlook to future directions concludes this review.     

In vitro inactivation of gonadotropin receptors,a membrane-associated action?

Summary Under in vitro conditions the time-dependent inactivation process of LH/HCG receptors is nearly identical in ovarian and testicular homogenates but different in gonadal membrane preparations. In the ovarian membranes the loss of binding sites during the first preincubation time is faster than in testicular membranes, especially in membranes of luteinized rat ovaries. Compared with the homogenates, however, the receptor-inactivation in the membranes is generally delayed. The inhibitory effect of metabolic agents on receptor inactivation indicates that membrane-associated actions are involved in this process.This work was supported in part by the Deutsche Forschungsgemeinschaft (Si 185/2). We thank Mrs Rita Rudolph for skilful technical assistance.     

In vitro inactivation of gonadotropin receptors, a membrane-associated action?

Under in vitro conditions the time-dependent inactivation process of LH/HCG receptors is nearly identical in ovarian and testicular homogenates but different in gonadal membrane preparations. In the ovarian membranes the loss of binding sites during the first preincubation time is faster than in testicular membranes, especially in membranes of luteinized rat ovaries. Compared with the homogenates, however, the receptor-inactivation in the membranes is generally delayed. The inhibitory effect of metabolic agents on receptor inactivation indicates that membrane-associated actions are involved in this process.     

Genetic and molecular analysis of the synaptotagmin family

Secretion is a fundamental cellular process used by all eukaryotes to insert proteins into the plasma membrane and transport signaling molecules and intravesicular proteins into the extracellular space. Secretion requires the fusion of two phospholipid bilayers within the cell, an energetically unfavorable process. A conserved repertoire of vesicle-trafficking proteins has evolved that function to overcome this energy barrier and temporally and spatially control membrane fusion within the cell. Within neurons, opening of synaptic calcium channels and subsequent calcium entry triggers synchronous synaptic vesicle exocytosis and neurotransmitter release into the synaptic cleft. After fusion, synaptic vesicles undergo endocytosis, are refilled with neurotransmitter, and return to the vesicle pool for further rounds of cycling. It is within this local synaptic trafficking pathway that the synaptotagmin family of calcium-binding synaptic vesicle proteins has been postulated to function. Here we review the current literature on the function of the synaptotagmin family and discuss the implications for synaptic transmission and membrane trafficking. Received 14 August 2000; received after revision 20 September 2000, accepted 14 October 2000     

Inhibition of diuresis in the tsetse fly (Glossina morsitans) by ouabain and acetazolamide.

Acetazolamide and ouabain, metabolic inhibitors which interfere with certain membrane transport systems, reduce the rate of water elimination by male Glossina morsitans morsitans. The results suggest that water is transported across membranes during diuresis and that a ouabain sensitive Na+K+ATPase and an acetazolamide-sensitive carbonic anhydrase are involved in diuresis.     

Entry of herpesviruses into mammalian cells

The mechanism that herpesviruses use to enter cells is one of the most complex viral entry mechanisms studied so far. This complexity seems to mount as new participants, both cellular receptors and viral glycoproteins, are identified. Recent structural work on entry glycoproteins gD and gB from herpes simplex virus (HSV) 1 has illuminated the functional roles of these glycoproteins in the process of entry. In doing so, it provided information on the mechanism of two critical steps of HSV entry: receptor-mediated activation and membrane fusion. Remarkably, it is becoming clear that herpesviruses have a lot in common with other, simpler viruses.     

Role of laminin-nidogen complexes in basement membrane formation during embryonic development

Laminin and nidogen (entactin) are major glycoprotein components of basement membranes. At least seven different isoforms of laminin have been identified. Laminin and nidogen form high affinity complexes in basement membranes by specific binding between the laminin γ1 chain and the G3 globule of nidogen. Additional interactions between nidogen and collagen IV, perlecan and other basement membrane components result in the formation of ternary complexes between these matrix components. Nidogen is highly susceptible to proteolytic cleavage, and binding to laminin protects nidogen from degradation. Nidogen is considered to have a crucial role as a link protein in the assembly of basement membranes. Basement membrane components are synthesized at high levels during tissue growth and development, and sites of morphogenesis correlate with localized remodelling of basement membranes. The formation of distinct basement membrane matrices in the developing embryo is influenced by the laminin isoforms produced and by whether laminin and nidogen are co-expressed and secreted as a complex or are produced by cooperation between two cell layers. The potential roles of laminin-nidogen complexes, cell-matrix interactions, and other intermolecular interactions within the matrix in basement membrane assembly and stability are discussed in this review.     

Intracellular sterol trafficking

Summary Sterols are acquired by cells either biosynthetically by the interaction of cytoplasmic and endoplasmic reticulum elements, or by endocytosis. The subcellular distribution of sterols, however, argues that sterols are trafficked quickly from sites of acquisition to target membranes, particularly the plasma membrane. The mechanisms mediating this movement might include aqueous diffusion, vesicles of either a unique pathway or of the protein secretory pathway, or carrier proteins. These mechanisms are discussed and the limited data concerning each are presented. Finally, a theory is proposed which describes how sterols and other membrane reinforcing molecules might have driven the evolution of intracellular membranes, thus establishing the dynamic membrane system of modern eukaryotes.